1. Field of the Invention
The present invention relates to a novel cyanine compound for fluorescently labeling various biomolecules and a method for preparing the same.
2. Description of the Related Art
Substitution groups in proteins that can be bound to reactive groups in dyes can be inferred from the structure of amino acids (basic units of proteins). For example, amino acid residues, more specifically, amino (—CH2CH2CH2CH2NH2) for lysine, thiol (—CH2SH) of cystein, imidazole amine of histidine
secondary aliphatic hydroxyl group (—CH2CH(OH)CH3) of threonine, primary aliphatic hydroxyl group (—CH2OH) of serine and phenol hydroxyl group
of tyrosine and the like may be mentioned. Also, reactive groups in dyes may be bound to n-terminal amino group (—COCHRNH2) in amino acid. In addition, reactive groups in dyes may be bound to biomolecules such as sugar, glycoprotein and antibodies.
Reactive groups used for dyes or molecules for labeling biomolecules known to date are classified depending on substitution groups bound to biomolecules and are also called trivial names.
The most common reactive groups bound to amine of protein molecules are ester and isothiocyanate, and the most common reactive group bound to thiol of protein molecules is maleimide and reactive groups bound to hydroxyl groups of protein molecules are as follows:

In addition to these reactive groups, numerous researchers and enterprises are designing reactive group intermediates exhibiting superior performance. Most intermediates exhibit short reaction time with biomolecules and superior bonding performance, but are unstable in an aqueous solution state and are vulnerable to heat and produce by-products, while leaving groups are cleaved after reaction.
Water-soluble fluorescent dyes are actively applied to the field of biology. In order to incorporate water-soluble fluorescent dyes into biomolecules, the water-soluble fluorescent dyes should not cause photo-bleaching and quenching under aqueous or hydrophilic solution conditions, have a high molecular extinction coefficient sufficient to absorb a great deal of light, be within 500 nm or higher of visible or near infrared rays far from the fluorescent range of biomolecules and be stable under various pH conditions. However, structures of dyes useful for labeling biomolecules are limited due to various conditions.
All dyes are not fluorescent. Researchers in a variety of fields have developed dyes having fluorescent chromophores. Representative examples of fluorophores known to date include anthranilate, 1-alkylthic isoindoles, pyrrolinones, bimanes, benzoxazole, benzimidazole, benzofuran, naphthalenes, coumarins, stilbenes, carbazoles, phenanthridine, anthracenes, acridines, fluoresceins, eosins, rhodamines, pyrenes, chrysenes and the like. Derivatives similar to these fluorophores are also researched. These fluorophores are incorporated into various reactive groups to be bound to biomolecules and are thus commercially available as various products.
It is noted that these various fluorescent dyes should exhibit strong fluorescence in a medium in which most biomolecules are present, that is, an aqueous solution, in order that the dyes exhibit fluorescence applicable to the field of biology. The most commonly used fluorescent dyes for such application are xanthene-based fluorescein and rhodamine, and polymethine-based cyanine.
Cyanine dyes were first applied to biomolecular-labeling by Dr. Waggoner's team in the Carnegie Mellon University near the end of the 1980's. Dr. Waggoner's team found that binding of cyanine dyes mainly used for cloth dying or optical recording media, into which reactive groups that can be linked to proteins are incorporated, to proteins, causes expression of fluorescence and then reported the following several articles.    [Document 1] Ernst, L. A., Gupta, R. K., Mujumdar, R. B., and Waggoner, A. S. (1989) Cyanine Dye Labeling Reagents for Sulfhydryl groups. Cytometry 10, 3-10.    [Document 2] Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., and Waggoner, A. S. (1989) Cyanine Dye Labeling Reagents containing Isothiocyanate groups. Cytometry 10, 11-19.    [Document 3] Southwick, P. L., Ernst, L. A., Tauriello, E. W., Stephen, R. P., Mujumdar, R. B., Mujumdar, S. R., Clever, H. A., and Waggoner, A. S. (1990) Cyanine Dye Labeling reagents—Carboxymethylindocyanine Succinimidyl Esters. Cytometry 11, 418-430.    [Document 4] Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., and Waggoner, A. S. (1993) Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters. Bioconjugate Chem. 4, 105-111.    [Document 5] Mujumdar, S. R., Mujumdar, R. B., Grant, C. M., and Waggoner, A. S. (1996) Cyanine-Labeling Reagents: Sulfobenzindocyanine Succinimidyl Esters. Bioconjugate Chem. 7, 356-36.
Then, for various applications, numerous researchers introduced protein nucleophiles, that is, amine, thiol and hydroxyl groups, and pigments and fluorescent dyes for labeling biomolecules, into which reactive groups bound to electrophiles, that is, aldehyde, ketone and carboxylic acid groups, are incorporated.
Generally, cyanine dyes exhibit optical and pH stability, have narrow absorption and emission wavelength ranges and are fluorescent in the range of 500 to 800 nm. This fluorescence range of cyanine dyes does not overlap with the self-fluorescence range of biomolecules, thus advantageously making it easy to analyze. In addition, cyanine dyes exhibit high molecular extinction coefficients, although there are slight differences therebetween depending on characteristics of solvent and solubility. The following Formulas represent a generic structure of cyanine dyes shown in the documents and basic structures of hetero compounds known as derivatives.

Most commercially available cyanine dyes have indole structures as hetero rings and succinimidyl ester as reactive groups. The Formula represented below is a representative structure of cyanine dyes, which are commercially available under the trade names Cy3, Cy5 and Cy7 from GE healthcare Co., Ltd.

Unlike cloth dyes requiring various colors, fluorescent dyes for labeling biomolecules having a wide fluorescence wavelength range are not necessarily preferable. This is the reason that wavelengths of equipment using or measuring fluorescence are limited. Unless novel fluorescence analysis methods or apparatuses are developed, optical equipment is improved, or performance thereof is suited for dyes, variations in chromogens or structures varying light-absorption or light-emission wavelength ranges are not significant in view of commercialization in the field of dyes for labeling biomolecules.
It is known that like dyes having different chromogens, cyanine dyes have polymethine as a chromogen, regardless of incorporation of reactive groups, and thus substantially maintain fluorescence properties and undergo almost no variation in light-absorption and light-emission wavelengths.
Cyanine-labeling dyes having succinimidyl ester, used for labeling biomolecules, are dyed in a carbonate or phosphate buffer solution. Generally, the buffer is used in a concentration of 0.1M and the reaction is carried out at room temperature.
A dye is dissolved in N,N-dimethylformamide (DMF) or N,N-dimethyl sulfoxide (DMSO). 1 mg of the dye is dissolved in 100 μl of solvent and the resulting solution is then aliquoted. The dye is used in an excess of 5- to 100-fold equivalents with respect to biomolecules to be stained because, although a protein molecule is used in an amount of one equivalent, the number of amino, hydroxyl or thiol groups targeted by the reaction is much greater. Dyes require higher aqueous solution stability, in order for the dyes to be permeated into complicated protein structures and thus react therewith. However, it is disadvantageous that succinimidyl ester cannot be stably maintained for a long period of time.